ABSTRACT
BACKGROUND: An increasing body of evidence implicates the resident gut microbiota as playing a critical role in type 2 diabetes (T2D) pathogenesis. We previously reported significant improvement in postprandial glucose control in human participants with T2D following 12-week administration of a 5-strain novel probiotic formulation ('WBF-011') in a double-blind, randomized, placebo controlled setting (NCT03893422). While the clinical endpoints were encouraging, additional exploratory measurements were needed in order to link the motivating mechanistic hypothesis - increased short-chain fatty acids - with markers of disease. RESULTS: Here we report targeted and untargeted metabolomic measurements on fasting plasma (n = 104) collected at baseline and end of intervention. Butyrate and ursodeoxycholate increased among participants randomized to WBF-011, along with compelling trends between butyrate and glycated haemoglobin (HbA1c). In vitro monoculture experiments demonstrated that the formulation's C. butyricum strain efficiently synthesizes ursodeoxycholate from the primary bile acid chenodeoxycholate during butyrogenic growth. Untargeted metabolomics also revealed coordinated decreases in intermediates of fatty acid oxidation and bilirubin, potential secondary signatures for metabolic improvement. Finally, improvement in HbA1c was limited almost entirely to participants not using sulfonylurea drugs. We show that these drugs can inhibit growth of formulation strains in vitro. CONCLUSION: To our knowledge, this is the first description of an increase in circulating butyrate or ursodeoxycholate following a probiotic intervention in humans with T2D, adding support for the possibility of a targeted microbiome-based approach to assist in the management of T2D. The efficient synthesis of UDCA by C. butyricum is also likely of interest to investigators of its use as a probiotic in other disease settings. The potential for inhibitory interaction between sulfonylurea drugs and gut microbiota should be considered carefully in the design of future studies.
Subject(s)
Butyrates/blood , Diabetes Mellitus, Type 2/drug therapy , Probiotics/therapeutic use , Ursodeoxycholic Acid/blood , Bile Acids and Salts/analysis , Bile Acids and Salts/blood , Bile Acids and Salts/metabolism , Blood Glucose/drug effects , Butyrates/analysis , Butyrates/metabolism , Clostridium butyricum/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/microbiology , Fatty Acids, Volatile/analysis , Fatty Acids, Volatile/blood , Fatty Acids, Volatile/metabolism , Feces/chemistry , Gastrointestinal Microbiome/drug effects , Glycated Hemoglobin/analysis , Humans , Metabolomics , Probiotics/metabolism , Sulfonylurea Compounds/therapeutic use , Ursodeoxycholic Acid/analysis , Ursodeoxycholic Acid/metabolismABSTRACT
The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.
Subject(s)
Archaea , Electrons , Anaerobiosis , Archaea/genetics , Archaea/metabolism , Genomics , Geologic Sediments/microbiology , Methane/metabolism , Oxidation-Reduction , Phylogeny , Sulfates/metabolismABSTRACT
Sulfate-coupled anaerobic oxidation of methane (AOM) is a major methane sink in marine sediments. Multiple lineages of anaerobic methanotrophic archaea (ANME) often coexist in sediments and catalyze this process syntrophically with sulfate-reducing bacteria (SRB), but the potential differences in ANME ecophysiology and mechanisms of syntrophy remain unresolved. A humic acid analog, anthraquinone 2,6-disulfonate (AQDS), could decouple archaeal methanotrophy from bacterial sulfate reduction and serve as the terminal electron acceptor for AOM (AQDS-coupled AOM). Here in sediment microcosm experiments, we examined variations in physiological response between two co-occurring ANME-2 families (ANME-2a and ANME-2c) and tested the hypothesis of sulfate respiration by ANME-2. Sulfate concentrations as low as 100 µM increased AQDS-coupled AOM nearly 2-fold matching the rates of sulfate-coupled AOM. However, the SRB partners remained inactive in microcosms with sulfate and AQDS and neither ANME-2 families respired sulfate, as shown by their cellular sulfur contents and anabolic activities measured using nanoscale secondary ion mass spectrometry. ANME-2a anabolic activity was significantly higher than ANME-2c, suggesting that ANME-2a was primarily responsible for the observed sulfate stimulation of AQDS-coupled AOM. Comparative transcriptomics showed significant upregulation of ANME-2a transcripts linked to multiple ABC transporters and downregulation of central carbon metabolism during AQDS-coupled AOM compared to sulfate-coupled AOM. Surprisingly, genes involved in sulfur anabolism were not differentially expressed during AQDS-coupled AOM with and without sulfate amendment. Collectively, this data indicates that ANME-2 archaea are incapable of respiring sulfate, but sulfate availability differentially stimulates the growth and AOM activity of different ANME lineages.
Subject(s)
Archaea , Sulfates , Anaerobiosis , Archaea/metabolism , Geologic Sediments/microbiology , Humans , Methane/metabolism , Oxidation-Reduction , Phylogeny , Sulfates/metabolismABSTRACT
Methanol is often considered as a non-competitive substrate for methanogenic archaea, but an increasing number of sulfate-reducing microorganisms (SRMs) have been reported to be capable of respiring with methanol as an electron donor. A better understanding of the fate of methanol in natural or artificial anaerobic systems thus requires knowledge of the methanol dissimilation by SRMs. In this study, we describe the growth kinetics and sulfur isotope effects of Desulfovibrio carbinolicus, a methanol-oxidizing sulfate-reducing deltaproteobacterium, together with its genome sequence and annotation. D. carbinolicus can grow with a series of alcohols from methanol to butanol. Compared to longer-chain alcohols, however, specific growth and respiration rates decrease by several fold with methanol as an electron donor. Larger sulfur isotope fractionation accompanies slowed growth kinetics, indicating low chemical potential at terminal reductive steps of respiration. In a medium containing both ethanol and methanol, D. carbinolicus does not consume methanol even after the cessation of growth on ethanol. Among the two known methanol dissimilatory systems, the genome of D. carbinolicus contains the genes coding for alcohol dehydrogenase but lacks enzymes analogous to methanol methyltransferase. We analyzed the genomes of 52 additional species of sulfate-reducing bacteria that have been tested for methanol oxidation. There is no apparent relationship between phylogeny and methanol metabolizing capacity, but most gram-negative methanol oxidizers grow poorly, and none carry homologs for methyltransferase (mtaB). Although the amount of available data is limited, it is notable that more than half of the known gram-positive methanol oxidizers have both enzymatic systems, showing enhanced growth relative to the SRMs containing only alcohol dehydrogenase genes. Thus, physiological, genomic, and sulfur isotopic results suggest that D. carbinolicus and close relatives have the ability to metabolize methanol but likely play a limited role in methanol degradation in most natural environments.
Subject(s)
Cell Respiration , Desulfovibrio/metabolism , Genome, Bacterial , Genomics/methods , Methanol/metabolism , Sulfur Isotopes/analysis , Desulfovibrio/genetics , Desulfovibrio/growth & development , Phylogeny , RNA, Ribosomal, 16SABSTRACT
Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F420-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments.
ABSTRACT
The anaerobic oxidation of methane by anaerobic methanotrophic (ANME) archaea in syntrophic partnership with deltaproteobacterial sulfate-reducing bacteria (SRB) is the primary mechanism for methane removal in ocean sediments. The mechanism of their syntrophy has been the subject of much research as traditional intermediate compounds, such as hydrogen and formate, failed to decouple the partners. Recent findings have indicated the potential for extracellular electron transfer from ANME archaea to SRB, though it is unclear how extracellular electrons are integrated into the metabolism of the SRB partner. We used metagenomics to reconstruct eight genomes from the globally distributed SEEP-SRB1 clade of ANME partner bacteria to determine what genomic features are required for syntrophy. The SEEP-SRB1 genomes contain large multiheme cytochromes that were not found in previously described free-living SRB and also lack periplasmic hydrogenases that may prevent an independent lifestyle without an extracellular source of electrons from ANME archaea. Metaproteomics revealed the expression of these cytochromes at in situ methane seep sediments from three sites along the Pacific coast of the United States. Phylogenetic analysis showed that these cytochromes appear to have been horizontally transferred from metal-respiring members of the Deltaproteobacteria such as Geobacter and may allow these syntrophic SRB to accept extracellular electrons in place of other chemical/organic electron donors.IMPORTANCE Some archaea, known as anaerobic methanotrophs, are capable of converting methane into carbon dioxide when they are growing syntopically with sulfate-reducing bacteria. This partnership is the primary mechanism for methane removal in ocean sediments; however, there is still much to learn about how this syntrophy works. Previous studies have failed to identify the metabolic intermediate, such as hydrogen or formate, that is passed between partners. However, recent analysis of methanotrophic archaea has suggested that the syntrophy is formed through direct electron transfer. In this research, we analyzed the genomes of multiple partner bacteria and showed that they also contain the genes necessary to perform extracellular electron transfer, which are absent in related bacteria that do not form syntrophic partnerships with anaerobic methanotrophs. This genomic evidence shows a possible mechanism for direct electron transfer from methanotrophic archaea into the metabolism of the partner bacteria.
Subject(s)
Archaea/genetics , Archaea/metabolism , Bacteria/genetics , Bacteria/metabolism , Methane/metabolism , Anaerobiosis , Electron Transport , Genome, Archaeal , Genome, Bacterial , Geologic Sediments/microbiology , Hydrogen/metabolism , Metagenomics , Oxidation-Reduction , Phylogeny , Sulfates/metabolismABSTRACT
Marine methane seep habitats represent an important control on the global flux of methane. Nucleotide-based meta-omics studies outline community-wide metabolic potential, but expression patterns of environmentally relevant proteins are poorly characterized. Proteomic stable isotope probing (proteomic SIP) provides additional information by characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities, offering enhanced detection through system-wide product integration. Here we applied proteomic SIP to (15)[Formula: see text] and CH4 amended seep sediment microcosms in an attempt to track protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 unique proteins were identified, 11% of which were (15)N-labeled. Consistent with the dominant anaerobic oxidation of methane (AOM) activity commonly observed in anoxic seep sediments, proteins associated with sulfate reduction and reverse methanogenesis-including the ANME-2 associated methylenetetrahydromethanopterin reductase (Mer)-were all observed to be actively synthesized ((15)N-enriched). Conversely, proteins affiliated with putative aerobic sulfur-oxidizing epsilon- and gammaproteobacteria showed a marked decrease over time in our anoxic sediment incubations. The abundance and phylogenetic range of (15)N-enriched methyl-coenzyme M reductase (Mcr) orthologs, many of which exhibited novel post-translational modifications, suggests that seep sediments provide niches for multiple organisms performing analogous metabolisms. In addition, 26 proteins of unknown function were consistently detected and actively expressed under conditions supporting AOM, suggesting that they play important roles in methane seep ecosystems. Stable isotope probing in environmental proteomics experiments provides a mechanism to determine protein durability and evaluate lineage-specific responses in complex microbial communities placed under environmentally relevant conditions. Our work here demonstrates the active synthesis of a metabolically specific minority of enzymes, revealing the surprising longevity of most proteins over the course of an extended incubation experiment in an established, slow-growing, methane-impacted environmental system.
ABSTRACT
Tenericutes are a unique class of bacteria that lack a cell wall and are typically parasites or commensals of eukaryotic hosts. Environmental 16S rDNA surveys have identified a number of tenericute clades in diverse environments, introducing the possibility that these Tenericutes may represent non-host-associated, free-living microorganisms. Metagenomic sequencing of deep-sea methane seep sediments resulted in the assembly of two genomes from a Tenericutes-affiliated clade currently known as 'NB1-n' (SILVA taxonomy) or 'RF3' (Greengenes taxonomy). Metabolic reconstruction revealed that, like cultured members of the Mollicutes, these 'NB1-n' representatives lack a tricarboxylic acid cycle and instead use anaerobic fermentation of simple sugars for substrate level phosphorylation. Notably, the genomes also contained a number of unique metabolic features including hydrogenases and a simplified electron transport chain containing an RNF complex, cytochrome bd oxidase and complex I. On the basis of the metabolic potential predicted from the annotated genomes, we devised an anaerobic enrichment media that stimulated the growth of these Tenericutes at 10 °C, resulting in a mixed culture where these organisms represented ~60% of the total cells by targeted fluorescence in situ hybridization (FISH). Visual identification by FISH confirmed these organisms were not directly associated with Eukaryotes and electron cryomicroscopy of cells in the enrichment culture confirmed an ultrastructure consistent with the defining phenotypic property of Tenericutes, with a single membrane and no cell wall. On the basis of their unique gene content, phylogenetic placement and ultrastructure, we propose these organisms represent a novel class within the Tenericutes, and suggest the names Candidatus 'Izimaplasma sp. HR1' and Candidatus 'Izimaplasma sp. HR2' for the two genome representatives.
Subject(s)
Methane/metabolism , Phylogeny , Seawater/microbiology , Tenericutes/genetics , DNA, Bacterial/genetics , Genome, Bacterial , Genomics , In Situ Hybridization, Fluorescence , Methane/analysis , RNA, Ribosomal, 16S/genetics , Seawater/analysis , Tenericutes/classification , Tenericutes/isolation & purification , Tenericutes/metabolismABSTRACT
Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory-scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high-stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter-enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.
Subject(s)
Bacterial Proteins/genetics , Betaproteobacteria/genetics , Betaproteobacteria/metabolism , Biofilms , Bioreactors/microbiology , Metagenomics/methods , Phosphorus/metabolism , Phylogeny , Proteomics , RNA, Ribosomal, 16S/genetics , Sewage/microbiologyABSTRACT
The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at https://github.com/dib-lab/khmer/.
ABSTRACT
Large-scale recovery of genomes from isolates, single cells, and metagenomic data has been made possible by advances in computational methods and substantial reductions in sequencing costs. Although this increasing breadth of draft genomes is providing key information regarding the evolutionary and functional diversity of microbial life, it has become impractical to finish all available reference genomes. Making robust biological inferences from draft genomes requires accurate estimates of their completeness and contamination. Current methods for assessing genome quality are ad hoc and generally make use of a limited number of "marker" genes conserved across all bacterial or archaeal genomes. Here we introduce CheckM, an automated method for assessing the quality of a genome using a broader set of marker genes specific to the position of a genome within a reference genome tree and information about the collocation of these genes. We demonstrate the effectiveness of CheckM using synthetic data and a wide range of isolate-, single-cell-, and metagenome-derived genomes. CheckM is shown to provide accurate estimates of genome completeness and contamination and to outperform existing approaches. Using CheckM, we identify a diverse range of errors currently impacting publicly available isolate genomes and demonstrate that genomes obtained from single cells and metagenomic data vary substantially in quality. In order to facilitate the use of draft genomes, we propose an objective measure of genome quality that can be used to select genomes suitable for specific gene- and genome-centric analyses of microbial communities.
Subject(s)
Genome, Microbial , Metagenome , Metagenomics/methodsABSTRACT
Enhanced biological phosphorus removal (EBPR) is an important industrial wastewater treatment process mediated by polyphosphate-accumulating organisms (PAOs). Members of the genus Candidatusâ Accumulibacter are one of the most extensively studied PAO as they are commonly enriched in lab-scale EBPR reactors. Members of different Accumulibacter clades are often enriched through changes in reactor process conditions; however, the two currently sequenced Accumulibacter genomes show extensive metabolic similarity. Here, we expand our understanding of Accumulibacter genomic diversity through recovery of eight population genomes using deep metagenomics, including seven from phylogenetic clades with no previously sequenced representative. Comparative genomic analysis revealed a core of shared genes involved primarily in carbon and phosphorus metabolism; however, each Accumulibacter genome also encoded a substantial number of unique genes (> 700 genes). A major difference between the Accumulibacter clades was the type of nitrate reductase encoded and the capacity to perform subsequent steps in denitrification. The Accumulibacter clade IIF genomes also contained acetaldehyde dehydrogenase that may allow ethanol to be used as carbon source. These differences in metabolism between Accumulibacter genomes provide a molecular basis for niche differentiation observed in lab-scale reactors and may offer new opportunities for process optimization.
Subject(s)
Betaproteobacteria/genetics , Betaproteobacteria/metabolism , Wastewater/chemistry , Water Purification/methods , Aldehyde Oxidoreductases/genetics , Betaproteobacteria/enzymology , Bioreactors , Carbon/metabolism , Denitrification/genetics , Denitrification/physiology , Ethanol/metabolism , Genetic Variation/genetics , Metagenomics , Nitrate Reductase/genetics , Nitrogen Fixation/physiology , Phosphorus/metabolism , Phylogeny , Polymorphism, Single Nucleotide , Polyphosphates/metabolismABSTRACT
Hydrothermal vents are an important contributor to marine biogeochemistry, producing large volumes of reduced fluids, gasses, and metals and housing unique, productive microbial and animal communities fueled by chemosynthesis. Methane is a common constituent of hydrothermal vent fluid and is frequently consumed at vent sites by methanotrophic bacteria that serve to control escape of this greenhouse gas into the atmosphere. Despite their ecological and geochemical importance, little is known about the ecophysiology of uncultured hydrothermal vent-associated methanotrophic bacteria. Using metagenomic binning techniques, we recovered and analyzed a near-complete genome from a novel gammaproteobacterial methanotroph (B42) associated with a white smoker chimney in the Southern Lau basin. B42 was the dominant methanotroph in the community, at â¼80x coverage, with only four others detected in the metagenome, all on low coverage contigs (7x-12x). Phylogenetic placement of B42 showed it is a member of the Methylothermaceae, a family currently represented by only one sequenced genome. Metabolic inferences based on the presence of known pathways in the genome showed that B42 possesses a branched respiratory chain with A- and B-family heme copper oxidases, cytochrome bd oxidase and a partial denitrification pathway. These genes could allow B42 to respire over a wide range of oxygen concentrations within the highly dynamic vent environment. Phylogenies of the denitrification genes revealed they are the result of separate horizontal gene transfer from other Proteobacteria and suggest that denitrification is a selective advantage in conditions where extremely low oxygen concentrations require all oxygen to be used for methane activation.
ABSTRACT
Molecular surveys of aphotic habitats have indicated the presence of major uncultured lineages phylogenetically classified as members of the Cyanobacteria. One of these lineages has recently been proposed as a nonphotosynthetic sister phylum to the Cyanobacteria, the Melainabacteria, based on recovery of population genomes from human gut and groundwater samples. Here, we expand the phylogenomic representation of the Melainabacteria through sequencing of six diverse population genomes from gut and bioreactor samples supporting the inference that this lineage is nonphotosynthetic, but not the assertion that they are strictly fermentative. We propose that the Melainabacteria is a class within the phylogenetically defined Cyanobacteria based on robust monophyly and shared ancestral traits with photosynthetic representatives. Our findings are consistent with theories that photosynthesis occurred late in the Cyanobacteria and involved extensive lateral gene transfer and extends the recognized functionality of members of this phylum.
Subject(s)
Bioreactors/microbiology , Cyanobacteria/genetics , Genome, Bacterial , Phascolarctidae/microbiology , Phylogeny , Animals , Biological Evolution , Cyanobacteria/classification , Cyanobacteria/isolation & purification , Cyanobacteria/metabolism , Feces/microbiology , Genetics, Population , Male , Photosynthesis , RNA, Ribosomal, 16S , Waste Disposal, Fluid/instrumentation , Waste Disposal, Fluid/methodsABSTRACT
Over the past decade, technological advances in whole genome amplification, microfluidics, flow sorting, and high-throughput sequencing have led to the development of single-cell genomics. Single-cell genomic approaches are typically applied to anonymous microbial cells with only morphology providing clues to their identity. However, targeted separation of microorganisms based on phylogenetic markers, such as the 16S rRNA gene, is beginning to emerge in the single-cell genomics field. Here, we describe an in-solution fluorescence in situ hybridization (FISH) protocol which can be combined with fluorescence-activated cell sorting (FACS) for separation of single cells or populations of interest from environmental samples. Sequencing of DNA obtained from sorted cells can be used for the recovery of draft quality genomes, and when performed in parallel with deep metagenomics, can be used to validate and further scaffold metagenomic assemblies. We illustrate in this chapter the feasibility of this FISH-FACS approach by describing the targeted recovery of a novel anaerobic methanotrophic archaeon.
Subject(s)
Flow Cytometry , Genetics, Population , In Situ Hybridization, Fluorescence/methods , Metagenomics/methods , Archaea/genetics , Genome , Microfluidics/methods , RNA, Ribosomal, 16S , Sequence Analysis, DNA , Single-Cell AnalysisABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR) constitute a bacterial and archaeal adaptive immune system that protect against bacteriophage (phage). Analysis of CRISPR loci reveals the history of phage infections and provides a direct link between phage and their hosts. All current tools for CRISPR identification have been developed to analyse completed genomes and are not well suited to the analysis of metagenomic data sets, where CRISPR loci are difficult to assemble owing to their repetitive structure and population heterogeneity. Here, we introduce a new algorithm, Crass, which is designed to identify and reconstruct CRISPR loci from raw metagenomic data without the need for assembly or prior knowledge of CRISPR in the data set. CRISPR in assembled data are often fragmented across many contigs/scaffolds and do not fully represent the population heterogeneity of CRISPR loci. Crass identified substantially more CRISPR in metagenomes previously analysed using assembly-based approaches. Using Crass, we were able to detect CRISPR that contained spacers with sequence homology to phage in the system, which would not have been identified using other approaches. The increased sensitivity, specificity and speed of Crass will facilitate comprehensive analysis of CRISPRs in metagenomic data sets, increasing our understanding of phage-host interactions and co-evolution within microbial communities.
Subject(s)
Algorithms , Inverted Repeat Sequences , Metagenomics/methods , Bacteriophages/genetics , Genome, Archaeal , Genome, BacterialABSTRACT
The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race.